Manuscript title: Differential subcellular and extracellular localisations of proteins required
for insulin-like growth factor- and extracellular matrix-induced signalling events in breast
cancer progression.
Journal name: Breast cancer research
Additional file 9: Semiquantitative evaluation of immunohistochemical immunoreactivity
and consolidation of TMA scoring.
A biomarker scoring form was displayed alongside each TMA core for completion. A
standard biomarker scoring form was used for all protein targets investigated. Modified
biomarker scoring forms were used for ER, PR and HER2 (scoring forms not shown).
Specific questions were compulsory. Completion of the scoring forms for each TMA core
was required prior to moving on to the next TMA core. Briefly, the TMA scorers were asked
to record: their initials; the type of tumour present (provided as a choice between normal
breast tissue, DCIS, primary tumour, metastatic tumour tissue from the LN and missing
tissue); the amount of tumour present (as a percentage); the quality of the IHC staining
(provided as a choice between yes or no); the percentage of cells with cytoplasmic staining
(as a percentage); the intensity of the cytoplasmic staining (provided as a choice between 0,
1, 2 or 3); the percentage of cells with nuclear staining (as a percentage); the intensity of the
nuclear staining (provided as a choice between 0, 1, 2 or 3); the percentage of cells with
membrane staining (as a percentage); the intensity of the membrane staining (provided as a
choice between 0, 1, 2 or 3); the percentage of stromal staining (as a percentage); and the
intensity of the stromal staining (provided as a choice between 0, 1, 2 or 3).
IHC TMA scoring data was evaluated semiquantitatively using five scoring methods. Details
on these scoring methods are listed below.
Presence: Data were expressed as the proportion of the total number of samples in which
protein immunoreactivity was present within the normal breast tissue, DCIS, primary tumour
or LN metastatic tumour tissue.
Intensity: Data were expressed as a score between 0 and 3, corresponding to the presence of
negative, weak, intermediate, or strong staining, respectively, within the normal breast tissue,
DCIS, primary tumour or LN metastatic tumour tissue.
Percentage: Data were expressed as the proportion of cells or stroma staining positively
within the normal breast tissue, DCIS, primary tumour or LN metastatic tumour tissue.
Percentage class: Data were expressed as scores between 1 and 6 (1 = 0-4%; 2 = 5-19%; 3 =
20-39%; 4 = 40-59%; 5 = 60-79%, and; 6 = 80-100%), corresponding to the proportion of
cells staining positively within the normal breast tissue, DCIS, primary tumour or LN
metastatic tumour tissue.
Quickscore (Q score): Data were expressed as scores between 0 and 18 using a multiplicative
Q score system (Detre et al. 1995); this involves determining the staining intensity in
addition to the proportion of cellular staining. The multiplicative method allows for a “no
staining” category.
The exported .csv files from Distiller were converted to Microsoft Excel™ spreadsheets for
each search performed. Each of the spreadsheet rows corresponded to a TMA core (with a
specific tissue type). The variables recorded in the scoring form and the clinico-pathological
data was represented as separate columns in the spreadsheet. Duplicate rows represented
TMA cores originating from the same tissue sample and placed in duplicate TMAs. If there
were multiple TMA scorers then duplication rows would be present for each TMA scorer. If a
specific tissue sample was placed into more than one set of duplicate TMAs than duplicate
rows would be present for each set of duplicate TMAs. These duplicate rows were
consolidated into one row (if possible); the method of consolidation for each variable is listed
below. For statistical data analysis purposes, the consolidated rows (each with a specific
tissue type) were split up into four rows, each representing the results for a specific cellular
localisation.
Initials: If there was only one TMA scorer, the initials of that scorer was recorded in the
consolidated row. If there was more than one person who scored the TMA slide for IHC
immunoreactivity for a particular protein than the initials of all of the TMA scorers were
recorded in the consolidated row.
The type of tumour present: If the type of tissue was the same across the duplicate rows then
that type of tissue was recorded in the consolidated row. If there was more than one type of
tissue then a consolidated row was required for each tissue type represented.
The amount of tumour present: A mean percentage was calculated based on the proportion
of the tissue in the TMA core which contained primary or LN metastatic tumour tissue.
The quality of the immunohistochemical immunoreactivity: TMA cores with unsatisfactory
staining were discarded, while TMA cores with satisfactory immunoreactivity were
consolidated (by the methods mentioned in this section).
The percentage of cells with nuclear, cytoplasmic and membrane immunoreactivity: A
mean percentage was calculated based on the proportion of cells, within the normal breast
tissue, DCIS, primary tumour or LN metastatic tumour tissue which contained nuclear,
cytoplasmic and/or membrane immunoreactivity.
The percentage of stromal immunoreactivity: A mean percentage was calculated based on
the proportion of the stromal cells and ECM (which will be referred to as the ‘stroma’),
surrounding the normal breast tissue, DCIS, primary tumour or LN metastatic tumour tissue
which contained immunoreactivity.
The intensity of the nuclear, cytoplasmic, membrane and stromal immunoreactivity: The
highest intensity score was recorded in the consolidated row.